1. Field of the Invention
The present invention relates to methods for isolation of recombinant proteins, and namely methods that involve the removal of DNAK during the isolation procedure.
2. General Background and State of the Art
Obtaining substantial amounts of pure protein is essential in innumerable biological studies and indispensable to the biochemical characterization of proteins. The ease of growth, well-characterized genetics, and the large number of tools for gene expression has long made Escherichia coli (E. Coli) the organism of choice for protein overproduction.
DNAK is an abundant protein (about 1% of the total protein of E. coli) that interacts with a wide range of newly synthesized polypeptides whereby it acts as a chaperone. Chaperone proteins assist in the proper folding of newly expressed proteins and assembly into oligomers and thus prevent protein aggregation through interaction. DNAK binding to other proteins occurs when DNAK is bound to ADP, and release these proteins when bound to ATP. DNAK is also a required factor to disaggregate preformed protein aggregates, and it participates in the degradation of damaged proteins through protease specific channels.
While important for protein production, DNAK contamination presents a significant problem in protein purification. DNAK is able to bind to many proteins that are not endogenous to the bacterial host strain. Moreover, DNAK contamination may prevent the separation of recombinant fusion protein production, impair analysis of unfolding-refolding experiments, and cause strong antigenic responses in rats and rabbits even when the chaperone is present in trace amounts, which thereby affects antibody production.
Some recombinant protein isolation methods utilize E. coli DNAK deletion strains in order to eliminate DNAK contamination, but only when DNAK is not required to improve the solubility and the quality of the isolated recombinant proteins. Still, these deletions strains have narrower ranges of permissive temperatures for growth and exhibit multiple cellular defects which may reduce the overall yield of recombinant protein as well as stability of the strain.
Other methods involving fusion protein production utilize software based algorithms that determine appropriate amino acids surrounding the putative DNAK binding site of the recombinant protein and alter the sequence in order to decrease putative affinity for DNAK. Upon purification of these fusion protein bound to resin, MgATP plus soluble denatured E. coli proteins are used to wash the protein prior to elution. However, such methods are effective in only a limited number of cases, and not generally for all fusion proteins. Also, they do not eliminate contamination of their isolated recombination proteins that inherently contain putative DNAK binding sites. In addition, reagents of this procedure agents are costly and some are not suitable for commercial or therapeutic use (e.g. glycerol).
Alternate methods employ co-chaperone proteins which bind to DNAK in any of the nucleotide bound states. The co-chaperone protein was histidine tagged at the N-terminus, and this fusion recombinant protein was isolated by a one-step purification with nickel affinity chromatography. Despite the ability to remove DNAK via this co-chaperone, the method does not completely eliminate the DNAK, and relies on the transformation of an additional recombinant protein which reduces cellular resources needed to express the target recombinant protein at higher levels.